A: no enzyme control, B and C: no compound 1 controls. Conditions common to all reactions: 50 mM Tris-acetate, 10 mM MgCl2, 0.5 mM pNPP, and 76.42 nM (2.5 ng/mL) MptpB (except for condition A). A: 0.1 mM compound 1, B and C: 50% DMSO in lieu of compound 1, D: 0.1 mM compound 1, F: 0.3 mM compound 1, G: 0.4 mM compound 1, H: 0.6 mM compound 1, 0.001 mM Orthovanadate
Reaction condition E absent from graph above because it was spilled.
A: no enzyme control, B and C: no compound 2 controls. Conditions common to all reactions: 50 mM Tris-acetate, 10 mM MgCl2, 0.5 mM pNPP, and 76.42 nM (2.5 ng/mL) MptpB (except for condition A). A: 0.1 mM compound 2, B and C: 50% DMSO in lieu of compound 2, D: 0.1 mM compound 2, E: 0.2 mM compound 2, F: 0.3 mM compound 2, G: 0.4 mM compound 2, H: 0.6 mM compound 2, 0.001 mM Orthovanadate
Week 14:
MptpB enzyme assay with pNPP as surrogate substrate
MptpB enzyme assay with pNPP as surrogate substrate
Week 13:
ICM screening of cb_306 against chain A of 2ZO5 (PDB ID of open conformation of MptpB)
Week 12:
MptpB enzyme assay with pNPP as surrogate substrate
MptpB after FPLC and viva spin, trial 1
trial 2
MptpB (induced after OD600 = 0.8 for 5hrs at 30 degrees Centigrade) post FPLC purification, trial 1
trial 2
Week 11:
Of the 30,000 ligands in the MW set, I have tested ligands 2,001-18,000. None of these ligands yielded higher fitness scores than the ligands we chose to order. The top two ligand scores obtained from the first 2,000 ligands (sceened last week) were 98.66 and 88.14. The top five scores obtained this week (ligands 2,001-18,000) are listed below.
Week 10:
Week 9:
Grew up 1.5L of LB + BL21 and most likely ruined the protein
Grew up 3L of LB + BL21 inducing after 0.8 OD600. This value was reached 1hr 40min after addition of overnight growth. Next time, will use less overnight growth in 1L LB samples in order to catch an OD600 of 0.3.
Week 8:
FPLC purified MptpB
Concentrated and obtained the following readings
Concentrated MptpB after FPLC (1st reading)
2nd reading
3rd reading
First inhibition assay
Week 7:
Out
Week 6:
Lysed two bacterial pellets
Purified 2 samples independently (approx. 15 mL each)
Ran a gel using the sample that had a concentration 0.19 mg/mL
Lane 1: Skipped, Lane 2: PageRuler Prestained Protein, Lane 3: Soluble Fraction, Lane 4: Flow through, Lane 5: Wash, Lane 6: Elution #1, Lane 7: Elution #2
Concentrated Elution #1 and Wash for FPLC purification. A significant amount of MptpB was lost in the wash step.
- Joshua - avoid putting links to files here. Show results in a table or in a screenshot instead. -- Dr. B
Week 5:
Worked on Virtual Screening
Week 4:
Worked on PyMol
Week 3:
Transformed BL21 cells using the good, midi prepped plasmid sample
Picked two colonies, one from each of two plates, and grew up two samples for 12 hr overnight
Began to grow up protein (two 550mL samples of BL21 cells + LB + Kan left on the counter for 1 hr)
Picked a colony from the plate that had 10 uL of transformed plasmid plated (colonies are bigger and more spread out as opposed to the plate that had 50 uL of plasmid plated) - trial 2 of the protein expression scale up
Pelleted two samples of BL21 bacteria (trial 1 samples had a wait period at room temp of 1 hr before IPTG was added and the trial 2 sample didn't have an exponential growth pattern)
Scaled up bacteria and grew up MptpB once again (trial three) using 1M K3PO4, 10% glycerol with LB + Kan for the overnight growth of one sample. For the second sample I re-suspended the bacteria in fresh LB + Kan after growing it up in LB + Kan (centrifuged BL21 cells at 6000g for 5 min). The 10% glycerol was used as an extra energy source and the K3PO4 was used as a buffer since the 50mL of LB from overnight growth becomes acidic. For one sample I simply re-suspended the bacteria in fresh LB.
Sample A: 1M K3PO4, 10% glycerol used; Sample B: BL21 pelleted and re-suspended in fresh LB
Week 2:
Transformed DH5 alpha cells using the plasmid sample containing my GOI after it's been mini prepped
Left plate: 50mL of transformed BL21 plated; Right plate: 10mL of transformed BL21 plated
Grew up transformed DH5 alpha cells and midi prepped them
Sent two samples to sequencing one of which had a 100% match with my GOI
Week 1:
Confirmed a 100% match for my GOI in pNIC-Bsa4
Good pNIC-Bsa4 + MptpB gene read after Midi Prep:
Subject: reverse sequence; Query: forward sequence (The forward sequence has one N at 805 and the reverse sequence has an N at 716. The reverse sequence also has an insertion two nucloetides before the start codon since it has 859 instead of 858 bp.)
Query: forward sequence + G replacing N determined from reverse sequence; Subject: GOI
Figure 7: Restriction Enzyme Digest and PCR: Lane 1: Skipped, Lane 2: 1kb DNA ladder, Lane 3: EcoRI digest, Lane 4: PvuII digest, Lane 5: EcoRI + PvuII digest, Lane 6: uncut plasmid, Lane 7: tube A PCR, Lane 8: tube B PCR, Lane 9: tube C PCR, Lane 10: tube D PCR
Figure 8: First attempt at PCR for RE cloning
Figure 9: RE cloning: Lane 1: Skipped, Lane 2: 100bp Ladder, Lane 3: 0.016ng pGFP amplified using VDS1 and 2, Lane 4: 0.16ng pGFP amplified using VDS1 and 2, Lane 5: 1.6ng pGFP amplified using VDS1 and 2, Lane 6: Skipped, Lane 7: No DNA, Lane 8: Skipped, Lane 9: 0.016ng pGFP amplified using M13For and Rev, Lane 10: 0.16ng pGFP amplified using M13For and Rev, Lane 11: 1.6ng pGFP amplified using M13For and Rev, Lane 12: No DNA
Figure 12: First attempt at PCR for pLIC sequencing vectors of pNIC-Bsa4
Figure 13: PCR for pLIC sequencing vectors of pNIC-Bsa4
Figure 16: PCR for pLIC sequencing vectors of pNIC-Bsa4
Figure 19: PCR for pNIC-Bsa4 cloning
Figure 22: Option B of previous protocol(single best condition scaled up to 50ul)
Figure 25: Lane 1: Skipped Lane 2: Ladder; Ruoyi Pu Lane 3: Primary PCR Lane 4: Secondary PCR; Jeanette C. Lane 5: Primary PCR Lane 6: Secondary PCR; Sadhana B. Lane 7: Primary PCR Lane 8: Secondary PCR; Josh B. Lane 9: Primary PCR Lane 10: Secondary PCR
Figure 28: Lane 1: 100bp ladder, Lane 2: PCR^2 product
The top band matches up with the secondary PCR product. However, there appears to be another smaller band. The forward and reverse primers were probably used to amplify both strands. I continued with the cloning procedure despite the fact that my PCR^2 product appears to contain two amplified sequences.
Figure 30: Lane 1:100bp ladder, Lane 2: Skipped, Lane 3: PCR^2 product after PCR cleanup
The lower band appears much brighter than the upper (desired) band after PCR cleanup. Some of the MPTPb coding sequence may have not been eluted properly.
The pNIC-Bsa4 fragments don't appear because I mistakenly used 2.25 ng when treating with BsaI rather than 2.25 ug, the amount called for in the protocol for pNIC-Bsa4 cloning.
Figure 31: Lane 1: Skipped, Lane 2: 100bp ladder, Lane 3: primary PCR product, Lanes 4-8: Secondary PCR w/regular primers, Lane 9: pNIC-Bsa4 after treatment with BsaI
Figure 34: PCR^2 for trial 2 of version 2 of cloning: Lane 1: 100bp ladder, Lane 2: Skipped, Lanes 3-6: PCR^2 product
Figure 37: Lane 1: Skipped, Lane 2: 100bp ladder, Lane 3: primary PCR product sample 1, Lanes 4-5: PCR^2 product (sample 1 of primary PCR product and designed primers used), Lane 6: primary PCR product sample 2, Lanes 7-8: (sample 2 of primary PCR product and designed primers used)
Joshua's Research12.09.11
Reaction condition E absent from graph above because it was spilled.
Week 14:
Week 13:
Week 12:
Week 11:
Week 10:
Week 9:
Week 8:
Week 7:
Week 6:
- Joshua - avoid putting links to files here. Show results in a table or in a screenshot instead. -- Dr. B
Week 5:
Week 4:
Week 3:
Week 2:
Week 1:
Good pNIC-Bsa4 + MptpB gene read after Midi Prep:
Transformation Efficiency:
The top band matches up with the secondary PCR product. However, there appears to be another smaller band. The forward and reverse primers were probably used to amplify both strands. I continued with the cloning procedure despite the fact that my PCR^2 product appears to contain two amplified sequences.
The lower band appears much brighter than the upper (desired) band after PCR cleanup. Some of the MPTPb coding sequence may have not been eluted properly.
The pNIC-Bsa4 fragments don't appear because I mistakenly used 2.25 ng when treating with BsaI rather than 2.25 ug, the amount called for in the protocol for pNIC-Bsa4 cloning.